The present invention relates to IL-6 antagonist peptides, isolatable from a peptide library through the two-hybrids system by their ability to bind to the intracellular domain of gp130 and containing at least 5 amino acids. In particular, such peptides comprise an amino acid sequence, which is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, as well as salts, functional derivatives, precursors and analogs thereof.
Another object of the present invention is to provide the peptide in substantially purified form, in order to be suitable for use in pharmaceutical compositions, as active ingredient, in pathologies that require IL-6 activity inhibition.
The Two Hybrid System (THS) is a method that uses transcriptional activity as a system to detect protein-protein interactions. A gene fusion is constructed to encode the DNA-binding domain of the yeast transcription factor GAL4 as a hybrid with any protein xe2x80x98Xxe2x80x99(usually a defined mammalian protein being the xe2x80x9cbaitxe2x80x9d binding target). An additional gene fusion construct will encode the transcription activation domain of GAL4 fused to any protein xe2x80x98Yxe2x80x99 (usually a library of diverse proteins, the xe2x80x9cfishxe2x80x9d) (Fields et al., 1994). Whenever an X-Y interaction does occur, it will bring the activation domain close to sites on the DNA recognised by the GAL4 DNA-binding domain, therefore resulting in the expression of a flanking reporter gene regulated by these DNA sites. The reporter genes commonly used include: 1) lacZ, which produces blue colonies on plates or filters containing X-Gal; and 2) His3, a yeast gene involved in histidine biosynthesis, required for growth of host yeast cells.
Recently, Fields and his team have used the THS for screening a library of random peptides, instead of a cDNA library, in order to find peptides capable of binding to the retinoblastoma protein (Rb) (Yang et al., 1995).
The receptor system for Interleukin-6 (IL-6) is composed of two distinct receptor xe2x80x9csubunitsxe2x80x9d designated gp80 and gp130 (see Hirano et al., 1994).
The IL6 type cytokines elicit their signal through receptors that share the gp130 protein. Upon ligand binding gp130 homo- or heterodimerizes with the LIF and OSM receptor, thereby activating associated JAK tyrosine kinases. The JAKs phosphorylate the signal transducer (gp130) and latent transcription factors of the STAT family (Signal Transducer and Activator of Transcription), like STAT1 and STAT3 in the case of IL-6. STAT factors dimerize, translocate to the nucleus and bind to enhancer elements of IL-6 responsive genes (Lxc3xcttiken et al., 1993).
Deletion analysis of the intracellular domain of gp130 has defined short stretches of amino acids known as box1 and box2 sufficient to impart both mitogenic activity and binding of JAK proteins (Vanderkuur et al., 1994): these activities were also observed when the binding sites of STATs were deleted. Therefore two functions can be ascribed to JAK kinases: 1) activation of STAT-mediated gene expression; 2) activation of STAT-independent mitogenic activity at least in some hematopoietic cells.
Additional kinases are known to associate with to the intracellular portion of gp130, such as Hck, Fes, Btk and Tec (Matsuda et al., 1995). However these interactions have not been elucidated at the molecular level. Moreover, Tanner et al. have demonstrated that the box1 domain of cytokine receptors is required but not sufficient for interaction with JAK kinases and have suggested that the box1 sequences cooperate with other cytoplasmic domain sequences to effect JAK kinase association (Tanner et al., 1995). Even the molecular counterpart on JAK kinases of box1 and box2 has not been defined.
Synthetic peptides that inhibit IL-6 activity have been described in the International Patent Application WO 97/13781 (YEDA), which relates to peptides derived from the gp80 protein.
As a target in the THS, we have analysed the intracellular portion of the human IL-6 receptor (gp130-ICD). This THS screening should therefore identify candidate peptides which are able to directly interact with gp130-ICD in a phosphorylation-independent manner. Phosphorylation independent-interactions with gp130-ICD are known to occur in the transduction of the signal triggered by IL-6 type cytokines. gp130-ICD interacting counterparts of this type include protein kinases of the JAK family (Darnell et al., 1994).
Therefore, the main object of the present invention is an IL-6 antagonist peptide, isolatable from a peptide library through the two-hybrids system by their ability to bind to the intracellular domain of gp130 and containing at least 5 amino acids. According to a preferred embodiment of the invention, such pepddes contain up to 30 amino acids, more preferably 5-20, most preferably 8-16.
According to the present invention the xe2x80x9cbaitxe2x80x9d (xe2x80x9cXxe2x80x9d) used in the THS screening is the intracellular domain (ICD) of the gp130 protein. Such domain corresponds to the region from amino acid at position 642 to amino acid at position 918 (Yamasaki K. et al., 1988) of the IL6-R (gp130). The xe2x80x9cfishxe2x80x9d in the THS screening is a library of random peptides. Such library can be any commercial library or can be produced xe2x80x9cin-housexe2x80x9d by known methods.
False positives arising from the above screening may be eliminated as described in the literature (Bartel et al., 1993).
According to a further preferred embodiment, such peptides comprise an amino acid sequence, which is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, as well as salts, functional derivatives, precursors and analogs thereof.
When, in SEQ ID NO: 1, Xaa3 is Gly and Xaa4 is Leu, the peptide of the invention comprises the amino acid sequence of SEQ ID NO: 2.
xe2x80x9cAnalogsxe2x80x9d, as used in the present application, means those peptides, in which one or more of the amino acids in the above sequences are changed without substantially affecting the IL-6 antagonist activity. In particular, preferred changes for analogs in accordance with the present invention are what are known as xe2x80x9cconservativexe2x80x9d substitutions. Conservative amino acid substitutions include amino acids replacements with synonymous amino acids within the same group, which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule, Grantham, Science, Vol. 185, pp. 862-864 (1974).
The synonymous amino acid groups are those defined in Table I. More preferably, the synonymous amino acid groups are those defined in Table II; and most preferably the synonymous amino acid groups are those defined in Table III.
The term xe2x80x9csaltsxe2x80x9d herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the peptides of the invention or analogs thereof Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulphuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. Of course, any such salts must have substantially similar activity to the peptides of the invention or its analogs.
The definition xe2x80x9cfunctional derivativesxe2x80x9d as herein used refers to derivatives which can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the terminal N- or C-groups according to known methods and are comprised in the invention when they are pharmaceutically acceptable i.e. when they do not destroy the protein activity or do not impart toxicity to the pharmaceutical compositions containing them. Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl- or aroyl-groups.
The xe2x80x9cprecursorsxe2x80x9d are compounds which are converted into the peptides of the invention in the human or animal body.
The xe2x80x9cIL-6 antagonist activityxe2x80x9d means ability to inhibit IL-6 activity by antagonizing the binding of IL-6 to its receptor and/or by interfering with the function of the receptor system which transduces, intracellularly, molecular signals leading to the gene activation in IL-6-dependent cells, such as, for example, myeloma cells. Therefore, such activity may be measured by any of the assays known in the art. Such assays include proliferation of murine plasmacytoma T1165 cells, growth inhibition of mouse M1 myeloid leukemia cells, or production of acute phase proteins from hepatoma cells.
The peptides of the invention may be prepared by any well known procedure in the art, such as solid phase synthesis or liquid phase synthesis. As a solid phase synthesis, for example, the amino acid corresponding to the C-terminus of the peptide to be synthesised is bound to a support which is insoluble in organic solvents, and by alternate repetition of reactions, one wherein amino acids with their xcex1-amino groups and side chain functional groups protected with appropriate protective groups are condensed one by one in order from the C-terminus to the N-terminus, and one where the amino acids bound to the resin or the protective group of the xcex1-amino groups of the peptides are released, the peptide chain is thus extended in this manner. Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used.
Typically used protective groups include tboc (t-butoxycarbonyl), Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh (4,4xe2x80x2-dimethoxydibenzhydryl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl) and Cl2-Bzl (2,6dichlorobenzyl) for the amino groups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl) for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).
After synthesis of the desired peptide, it is subjected to the de-protection reaction and cut out from the solid support. Such peptide cutting reaction may be carried with hydrogen fluoride or trifluoromethane sulfonic acid for the Boc method, and with TFA for the Fmoc method.
The crude peptide thus obtained is then subjected to purification. Purification is carried out by any one of the methods known for this purpose, i.e. any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like. For example, HPLC (high performance liquid chromatography) can be used. The elution can be carried using a water-acetonitrile-based solvent commonly employed for protein purification.
Another object of the present invention is, therefore, to provide the peptide in substantially purified form, in order to be suitable for use in pharmaceutical compositions, as active ingredient, in pathologies that require IL6 activity inhibition.
Pathologies in which the new peptides according to the invention are advantageously used for prophylactic, therapeutic or diagnostic uses include haematological diseases, immune system diseases, bone diseases, tumours and auto-immune diseases, as well as therapy for transplantation including solid organ and cellular transplants.
Specific examples of the above categories include the following diseases: chronic lymphocytic leukaemia (CLL), plasmacytoma/multiple myeloma, Castleman""s disease (CD), osteoporosis, psoriasis, multiple sclerosis, lupus erithematosus, diabetes, rheumatoid arthritis as well as anaemia and wasting in chronic diseases.
Further objects and advantages of the invention will be evident in the following description.
An embodiment of the invention is the administration of a pharmacologically active amount of the peptide of the invention to subjects at risk of developing. pathologies requiring IL-6 activity inhibition or to subjects already showing such pathologies.
Any route of administration compatible with the active principle can be used, but particularly preferred is the parenteral administration because it permits to have, in short times, systemic effects.
The dose of peptide to be administered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient.
The pharmaceutical composition for parenteral use can be prepared in injectable form comprising the active principle and a suitable vehicle. Vehicles for the parenteral administration are well known in the art and comprise, for example, water, saline solution and physiologic buffers. The vehicle can contain smaller amounts of excipients in order to maintain the solution stability and isotonicity.
The preparation of the cited solutions can be carried out according to the ordinary modalities.
The present invention has been described with reference to the specific embodiments, but the content of the description comprises all modifications and substitutions which can be brought by a person skilled in the art without extending beyond the meaning and purpose of the claims.
The invention will now be described by means of the following Examples, which should not be construed as in any way limiting the present invention. The Examples will refer to the Figures specified here below.